Enterohemorrhagic E. coli (EHEC) serotype O157:H7, an important agent of diarrheal disease, triggers the formation of filamentous actin pedestals on intestinal epithelial cells beneath sites of bacterial attachment. The ability to generate actin pedestals promotes late stage intestinal colonization and permits the formation of large aggregates on the epithelial surface. To generate pedestals, EHEC injects two effectors, Tir and EspFU, into mammalian cells via a type III secretion system. Tir is inserted into the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. The C-terminal cytoplasmic domain of Tir is recognized by IRTKS (Insulin Receptor Tyrosine Kinase Substrate), a mammalian adaptor/effector that promotes the formation of F-actin and protrusive membrane structures at the plasma membrane. IRTKS also binds PI(4,5)P2 and deforms membranes, and binds the GTPase Rac, which is also known to stimulate actin assembly. Importantly, a C-terminal IRTKS SH3 domain binds to EspFU,, potentially linking it to Tir. EspFU contains multiple 47-residue proline-rich repeats and activates the actin nucleation promoting factor (NPF) N- WASP by mimicking and displacing an autoinhibitory N-WASP peptide. N-WASP is required for efficient translocation of Tir and EspFU, but if this block is overcome, EspFU can trigger an N-WASP-independent pathway for actin assembly, presumably by interacting with an alternative mammalian actin NPF. These findings suggest a model in which host actin assembly initially promotes translocation of Tir and EspFU, both of which bind IRTKS to assemble a complex at the plasma membrane, clustered by interaction with bacterial intimin, that potently stimulates two pathways of actin assembly. Tir/EspFU-mediated actin assembly may in turn promote more efficient type III translocation, and, by unknown means, epithelial colonization in vivo. IRTKS may play a role in pedestal formation in addition to linking Tir to EspFU, since pilot experiments suggest that the IRTKS binding sequence of EspFU enhances pedestal formation even when EspFU is directly fused to Tir. The following aims will be pursued to investigate both N-WASP- dependent and -independent mechanisms of actin pedestal formation, and to examine potential roles of pedestal formation during mammalian infection: (1) Determine whether Tir-, EspFU-, PI(4,5)P2-, and/or Rac-binding activity is important for IRTKS to promote actin pedestal formation; (2) Identify mutants of EspFU that are defective for the N-WASP-dependent and/or -independent pathways of pedestal formation; (3) Determine the relative importance of the N-WASP-dependent and N-WASP- independent pathways in pedestal formation on polarized intestinal epithelial cells, and (4) Investigate whether pedestal formation promotes stable bacterial attachment, disruption of tight junctions and/or translocation in vitro, and the clonal expansion of microcolonies on intestinal epithelium during infection.